Performance of ProLong antifade reagents for fixed cells

ProLong Gold antifade reagent performance

ProLong Gold antifade reagent has become a widely used antifade reagent for Molecular Probes™ high-performance fluorescent dyes and stains because it preserves signal across the entire visible spectrum and causes little or no quenching of the initial signal. ProLong Gold is room-temperature stable—keep it on the bench, ready to use in your next imaging experiment.

ProLong Diamond antifade reagent performance

ProLong Diamond antifade reagent delivers all the performance of ProLong Gold reagent with Alexa Fluor™ dyes and provides additional protection for traditional dyes and fluorescent proteins. ProLong Diamond reagent is a superior antifade and mountant, offering protection across the visible spectrum. Keep ProLong Diamond reagent refrigerated for the best performance.

A 60-second time-lapse showing the enhanced resistance to photobleaching afforded by ProLong antifade reagents. Fixed HeLa cells were labeled with fluorescein phalloidin and mounted in ProLong Diamond reagent, ProLong Gold reagent, or 50% PBS/glycerol. Images were acquired at 12-second intervals using a 20x objective with continuous illumination from a standard 100-watt Hg-arc lamp.

Photobleaching resistance for fluorescent dyes and proteins with ProLong antifade reagents. Photobleaching resistance was quantified on a LSM 710 (Zeiss) confocal microscope. HeLa or U2OS cells were stained and mounted using standard ICC protocols. Five regions within three fields of view were scanned 50x with a 1.58 μs dwell time per pixel. Excitation wavelength and intensity were optimized for the fluorophore being measured.

Find the right ProLong antifade reagent for fixed cells

Performance across the spectrum

ProLong Diamond reagent is a superior antifade and mountant, offering protection across the visible spectrum and exhibiting less initial quenching of the fluorescent signal. It provides additional protection for traditional dyes such as FITC and fluorescent proteins such as GFP. In a comparison of commercial hard-mount antifade reagents in a standardized experiment, ProLong mountants outperformed other products across the analysis spectrum.

Comparison of degree of photobleaching protection by ProLong antifades for various fluorescent dyes

Fluorescent dye

Ex/Em (nm)

Resistance to photobleaching*

ProLong Gold

ProLong Diamond

VECTASHIELD™ HardSet
Mounting Medium

Hoechst™

350/461

100%

100%

90%

DAPI

345/455

90%

90%

90%

BODIPY™ FL

505/513

70%

100%

50%

Alexa Fluor 488

495/519

80%

100%

90%

GFP

488/510

40%

90%

90%

Fluorescein

494/518

80%

90%

90%

Cy®3

550/570

80%

80%

80%

Alexa Fluor 546

556/575

100%

100%

100%

Tetramethylrhodamine

555/580

80%

80%

80%

Alexa Fluor 555

555/565

90%

100%

90%

Alexa Fluor 568

578/603

70%

90%

90%

Texas Red™

595/615

90%

90%

90%

Alexa Fluor 594

590/617

100%

100%

90%

TO-PRO™-3

642/661

90%

90%

90%

Alexa Fluor 647

652/668

100%

90%

0%

Cy®5

650/670

100%

100%

90%

*The degree of photobleaching protection is compiled from a combination of bright field exposure and confocal scanning data to represent typical user experience in fluorescence imaging applications.VECTASHIELD is a trademark of Vector Laboratories, Inc., Burlingame, CA. Hoechst is a trademark of Hoechst, GmbH. Cy is a trademark of GE Healthcare UK, Ltd.

Green chemistry

For added convenience and a reduction in the environmental impact associated with cold storage requirements, our antifade reagents are provided in dropper bottles with storage right at your bench. In addition, these reagents are formulated with reduced sodium azide to help minimize environmental and health hazard exposure risks. Learn more about these green chemistry initiatives